Alternative ligands for measurement and purification of ecdysteroid receptors in Drosophila Kc cells.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 25-33 (1986) Alternative Ligands for Measurement and Purification of Ecdysteroid Receptors in Drosophila K, Cells Becky A. Sage, Denis H.S. Horn, Thomas M. Landon, and John D. O’Connor Department of Biology, University of California, Los Angeles (B.A.S., ‘EM.L., J. D.O.); Division of Applied Organic Chemistry, C.S.I.R.O., Melbourne, Victoriu, Australia (D.H.S.H.) An ecdysteroid-binding protein has been demonstrated in nuclear and cytosolic extracts of cultured Drosophila cells (K, cells). Attempts t o purify the binding moiety have been hampered because of the small number of binding sites (ca 1,00O/celI) and sensitivity of the ecdysteroid binding moiety and toward salt (dissociation at 150 mM). Recently 3~~[~H]kaladasterone 3~x[~H]muristerone A have been synthesized. The binding kinetics of these two ecdysteroids are similar t o ponasterone A. Photoaffinity labeling of the ecdysteroid receptor in Kc cells was attempted using the 3~x[~H]kaIadasterone, and standard protein chromatographic techniques have been used t o examine the 3~~[~H]muristerone A-receptor complex, which is less sensitive to salt dissociation. Attempts t o characterize the protein t o which these ligands have been attached will be discussed. Key words: photoaffinity labeling, kaladasterone, muristerone A INTRODUCTION For the past several years, the & cell line of Drosophila melunogaster, established by Echalier and Ohanessian [l], has been used as a model system to study ecdysteroid action. There are four particular advantages inherent in using an established insect cell line to study ecdysteroid action. The first is the availability of large quantities of biological material. Two additional advantages are that the & cells do not metabolize ecdysteroids, nor do they contain any endogenous hormone. This means that one can expose the & cells to ecdysteroids of known concentration and have the dosage of hor- Acknowledgments: We thank Dr. Maria A. Tanis for her efforts in synthesizing the precursor for the radioligand, [3H]ponasteroneA. This work was supported by a grant from the National Science Foundation. Address reprint requests to Dr. John D. O’Connor, Department of Biology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024. 0 1986 Alan R. Liss, Inc. 26 Sageetal mone remain constant over a period of days. A final advantage of the K, cell line is that the cells do show characteristic responses when exposed to physiological concentrations of ecdysteroids. These responses have been documented elsewhere, and include G2 arrest 121, changes in morphology ,increased intercellular adhesion ,and induction of acetylcholine esterase , dopa decarboxylase , and ecdysteroid-inducible polypeptides 18. Such responses are associated with the presence of an intracellular binding moiety or ecdysteroid receptor. CHARACTERIZATIONOF ECDYSTEROID RECEPTORS IN DROSOPHILA K, CELLS In addition to its association with biological responses, an intracellular binding protein must fulfill three kinetic criteria: Binding must be saturable, specific, and of high affinity. The characterization of these three criteria for the ecdysteroid receptor present in K, cells has been previously documented .Using a radioligand of high specific activity ([3H]ponasteroneA, structure C, Fig. 1, SA* 106 Cilmmol), an ecdysteroid binding moiety was obtained from both cytosolic and nuclear preparations of K, cells not previously exposed to hormone. This binding moiety demonstrated saturability, and specificity for ponasterone A (Kd 3 nM), 20-OH-ecdysone (Kd 200 nM), and ecdysone (Kd > 10 pM). These affinities correlated precisely with the ecdysteroids' respective biological activities and, in the case of the latter two ligands, with their physiological concentrations. Similar data have been obtained for imaginal discs . - - - @o;o#o" HO yH HO bH HO # 0 HO OH HO 0 (I) (II) Ho& bH OH HO HO 0 (m) Ho 0 (m) Fig. 1. Ecdysteroid structures. I, ecdysone; 11, 20-OH-ecdysone, the molting hormone; Ill, ponasterone A; IV, rnuristerone A. *Abbreviations: HPLC = high-performance liquid chromatography; Kd = dissociation constant; SA = specific activity. Purification of Ecdysteroid Receptors 27 When the hydrodynamic characteristics of this ecdysteroid receptor were examined, it was found that the binding moiety present in both cytosolic and nuclear preparations of K, cells sediments at 6 S. A 4 S sedimentation value could also be obtained for the cytosolic receptor, but this occurred only if the cells were frozen at -20°C prior to their homogenization and the preparation of a high-speed cytosol. The presence of the ecdysteroid binding moiety in both cytosolic and nuclear preparations suggests that the ecdysteroid receptor is not specifically localized within K, cells. Moreover, the data indicate that there exists within K, cells a resident population of nuclear receptors whose number does not significantly increase upon exposure to hormone . The existence of a significant population of resident nuclear receptors has also been documented in imaginal discs [lo], where approximately 95% of the receptor isolated from third instar imaginal discs is localized in the nuclei of the imaginal disc cells. This nuclear localization of the ecdysteroid receptor in imaginal discs might represent its normal or regular residence or the translocation of receptor into disc nuclei by previous exposure to hormone. Support for the former hypothesis has been presented previously . This observation of the nuclear localization of the ecdysteroid receptor in hormonally naive cells leads one to propose the revised paradigm for ecdysteroid action shown in Figure 2. Briefly, after 20-OH-ecdysone enters the cell, it enters the nucleus and binds to a receptor. This hormone-receptor complex then interacts with the DNA to induce new RNA transcripts. The hormone might also bind to receptors present in the cytosol, but the exact role of these complexes has not as yet been identified. e HO pH HO& I Processing 'bH HO 0 20-OH-Ecdysone \ Induced Prolein Synthesis Fig. 2. Paradigm of ecdysteroid mode of action. The arthropod molting hormone, 20-OHecdysone (E), enters the target cell by diffusion and travels into the nucleus where it binds to a receptor molecule (R). In the nucleus, the ecdysteroid receptor complex interacts with the DNA to induce new RNA transcripts. Ecdysteroid receptor molecules may also be present in the cytosol (stippled R) and able t o bind free ecdysteroid. Functions for these cytosolic ecdysteroid receptor complexes have not as yet been clearly identified. 28 Sageetal PURIFICATION OF THE ECDYSTEROID RECEPTORS IN DROSOPHILA K, CELLS To understand fully the mode of action of the ecdysteroid-receptor complex, one must have the ecdysteroid receptor purified. Several difficulties arise as one attempts to use standard purification techniques for this protein binding moiety. The first difficulty is the fact that ecdysteroid receptor concentrations are very small. It is estimated that there are 1,000-1,500 receptorslcell. This receptor number is tenfold less than that found in most other steroid systems [11,12]. The second difficulty is that the binding activity of unloaded receptor is inherently labile when subjected to standard protein purification techniques. Therefore, the ecdysteroid receptor needs to remain loaded with hormone during most manipulations. The fact that the steroid hormone complex has a relatively rapid dissociation constant at both 4°C and 22°C [S] makes the necessity for constant loading of the receptor even more difficult to maintain. A final difficulty, unique to the ecdysteroid system, is that the binding of the ecdysteroid to its receptor is salt-sensitive. Using [3H]ponasteroneA as a radioligand, >50% of the bound ligand dissociates during a 30-min incubation with 150 mM salt at 24°C. Such rapid dissociation in the presence of relatively low salt concentrations precludes the use of standard ion exchange chromatography techniques and HPLC protein separations that require high salt buffers. Our laboratory has tried to circumvent these difficulties by attempting to link covalently a radiolabeled ecdysteroid to its receptor by means of photoaffinity labeling. Although the conjugated 7-ene-6-one system, common to the ecdysteroids (absorption maximum 242 nm), is photoreactive, we have been unable to demonstrate significant covalent binding of either ponasterone A, 20-OH-ecdysone, or muristerone A (see Fig. 1for structures) to the ecdysteroid receptor. Similar difficulties have also been documented for other steroid receptor systems [13,14]. However, it has been found that steroid ligands, which contain a conjugated diene-one functional group, are generally more photoreactive [14,15]. This enhanced photoreactivity can be attributed partially to the fact that the absorption maximum of the conjugated diene-one system is 300 nm. This means that one should be able to irradiate ecdysteroid receptor preparations more effectively using wavelengths of light >320 nm. In doing so, one decreases the possibility that the protein population will undergo photodegradation during the photolysis procedure. One biologically active ecdysteroid that contains this conjugated dieneone system is kaladasterone . When unlabeled kaladasterone is incubated with a nuclear receptor preparation obtained from K, cells [S], the data shown in Figure 3 are obtained. The specific, rapid photoinactivation of receptor binding activity during UV irradiation of the kaladasterone receptor complex can be explained in one of two ways. Either the irradiation is causing the kaladasterone to become bound covalently to the receptor or the irradiation causes kaladasterone to destroy the ability of the receptor to bind the radioligand, [3H]ponasteroneA, following the irradiation procedure. To determine which of these events is occurring, the kaladasterone must be radiolabeled. A protocol for the synthesis of 3a[3H]kaladasterone from - - Purificationof Ecdysteroid Receptors 10 20 30 40 50 29 60 uv Irradiation Time, minutes Fig. 3. Photoinactivation of K, ecdysteroid binding moieties. Nuclear ecdysteroid receptor preparations  were incubated with saturating amounts of unlabeled 20-OH-ecdysone (1 pM) or kaladasterone (100 nM). Receptor preparations were then irradiated for given periods of time (Osram HBO-200W mercury lamp with glass filter, h > 320 nm, 4OC). Aliquots of the irradiated receptor preparations were then incubated with high salt (KCI concentration 400 mM, 30 rnin, 24OC) to dissociate any noncovalent binding of ecdysteroid to the receptor. The samples were then centrifuged through Sephadex C-25 to remove free ecdysteroid and the KCI. The degree of photoinactivation of the ecdysteroid receptor was measured by labeling the samples with [3H]ponasteroneA (60 min, 24OC, 1 nM) and determining bound ligand using a dextran-coated charcoal assay . Ecdysteroids present during photolysis: 0, 20-OH-ecdysone; A,kaladasterone; 0 ,control, no ecdysteroids present. - muristerone A was developed by one of us (D.H.S.H.) and is briefly outlined in Figure 4. Following the final step of mild base hydrolysis, the [3H]kaladasterone was purified on reverse-phase HPLC (Waters RCM-100, Radial-Pak CI8 pBondapak lop; 55% methanol in water; 2.0 mllmin; elution time of kaladasterone standard was 6 min 45 sec) and its specific activity determined to be -4 Cilmmol. Saturation kinetics of a nuclear receptor preparation incubated with [3H]kaladasteroneare shown in Figure 5. The Kd from the Scatchard [17l analysis of these data was calculated to be 15.0 nh4. In addition, the kaladasterone was shown to have biological activity in the Kc cell line by inducing a G2 arrest in the cell cycle. Irradiation of a [3H]kaladasterone nuclear receptor preparation initially resulted in the photodegradation of the ecdysteroid receptor’s binding capability. The only condition that was effective in reversing this destruction of receptor was to perform the photolysis under a nitrogen atmosphere. Under a nitrogen atmosphere, radioactivity was found associated with TCA-precipitable protein (Table 1).However, if these proteins are separated using HPLC anion exchange chromatography, the results shown in Figure 6 are obtained. The absorbance and radioactivity profiles of the eluting proteins show that the [3H]kaladasterone is being covalently cross-linked to a number of different protein species. Similar results of nonspecific photoaffinity labeling are 30 Sageetal HO 0" HO Muristerone A HO OH 3-Keto-Muristerone A HO 10% Aqueous CH,COOH HO [3~x-~H]-Muristerone A (-64% y i e l d ) HO OH 4 [3~x-~H]-KoIadasterone 16 - 08 48- 04 N 0 0 32c 3 0 m - __--_--_*--- I 5 20 40 l l 60 , l 80 1 I * 100 3H-Kaladosterone. nM Fig. 4. Outline of the syntheses of 3c~[~H]rnuristerone A and 3c~[~H]kaladasterone from rnuristerone A. Fig. 5. [3H]Kaladasteronesaturation of nuclear receptor preparation from K, cells. Aliquots of a Kc nuclear extract  were incubated with increasing concentrations of [3H]kaladasterone (75 rnin, 24OC). Bound ligand was determined by duplicate dextran-coated charcoal assays . Nuclear receptor preparations were incubated with either [3H]kaladasterone ( 0 )or [3H]kaladasterone with 100-fold excess ponasterone A (A). Saturable binding of [3H]kaladasteronewas obtained by subtraction of these two curves (0).Inset shows Scatchard [I71 analysis of saturation binding data. Purification of Ecdysteroid Receptors I 60 8 16 31 24 Time ( m i d Fig. 6. HPLC separation of photoaffinity labeled proteins present in a K, nuclear receptor preparation. A K, nuclear extract, in 50 mM potassium phosphate buffer, was incubated with [3H]kaladasterone (50 nM), placed into a glass reaction vessel, and equilibrated with nitrogen gas for 20 min. The sample was then photolyzed for 20 min under an N2 atmosphere (CanradHanovia medium pressure, mercury-vapor lamp, 450 W; h > 320 nm; 4OC). A portion of the sample was then analyzed by DEAE anion exchange HPLC chromatography. (Chromatographic conditions: Pharmacia Mono Q HR 5/5 anion exchange column; 1 mllmin; elution buffer at time 0 was 50 mM Tris, pH 8.0, followed at 3 rnin by a 10 min gradient of 0 to 350 m M KCI in 50 mM Tris, and at 18 min by 1.0 M KCI in 50 m M Tris.) Absorbance was monitored Fractions of 0.5 ml were collected and monitored for radioactivity (0-0). at 280 n m (-). TABLE 1. Photoaffinity Labeling of a Nuclear Extract From Kc Cells K, cell linea Radioligandb &-WT K,-WT K,-WT K,-BRK,-WT &-WT [3H]KAL [3H]KALC r3H]KAL [3H]KAL [3H]PNA [3H]PNAC Nitrogen equilibration TCA-precipitable protein (dpmi100 fig) + + + + + 2,540 1,230 170 620 680 260 '&-WT designates a receptor-positive cell line; K,-BR - designates a receptor-negative cell line. bRadioligand abbreviations: [3H]KAL is [3H]kaladasterone, [3H]PNA is [3H]ponasterone A. 'Experiments in which excess radioligand was removed prior to irradiation procedure. also observed when free kaladasterone is removed from the nuclear receptor preparation prior to the irradiation procedure. These data indicate that [3H]kaladasterone is a possible photoaffinity ligand, but the high degree of nonspecific covalent binding precludes using the ligand on a crude receptor preparation. The synthesis of radiolabeled [3H]kaladasteronealso provided our laboraA. In early experitory with an additional radioligand, 3~t[~H]muristerone ments with muristerone A, it was discovered that this particular ligand, 32 Sageetal 800 700 - 600 C 0 ._ ; ;500P LL \ E 400- U Q 200 100 - Fig. 7. HPLC anion exchange chromatography of K, 4 S cytosol receptor preparation. Cytosol receptor was obtained from frozen cells  and precipitated with 33.33% ammonium sulfate. The protein pellet was resuspended in 10 rnM Tris buffer, pH 7.3, and incubated with [3H]muristerone A (30 nM). An aliquot of the radiolabeled receptor preparation was then subjected to DEAE anion exchange HPLC chromatography. (Chromatographic conditions: Pharmacia Mono Q HR 515 anion exchange column; 1 ml/rnin; time 0 elution buffer was 50 rnM Tris, pH 8.0, followed at 2 rnin by an 11 rnin 0 to 400 m M KCI gradient in 50 rnM Tris and at 17 rnin by final elution with 1.0 M KCI in 50 rnM Tris.) Absorbance was monitored at 280 nm (-). Fractions of 0.5 m l were collected and counted for radioactivity (x-x). when bound to the ecdysteroid receptor, was much less sensitive to dissociation with high salt (data not shown). In addition, saturation studies, using [3H]muristerone A as the radioligand, indicate that muristerone A has an affinity for the ecdysteroid receptor (Kd 4 nM) that is similar to ponasterone A. Therefore, current efforts in our laboratory are being directed toward using the [3H]muristerone A as a radioligand to monitor receptor binding activity while attempting protein separation techniques that utilize high salt conditions. The data shown in Figure 7 document one such attempt of an HPLC anion exchan e separation of the 4 S form of the K, cell cytosol receptor. Using the [ Hlmuristerone A, it now seems probable that a 10,000fold purification of the ecdysteroid receptor can be achieved and subsequent monoclonal antibodies to the receptor generated. - F LITERATURE CITED 1. Echalier G, Ohanessian A: Isolement en cultures in vitro, de lignees cellulaires diploides de Drosophilu rnelunogaster. CR Acad Sci Series D 268, 1771 (1969). Purification of Ecdysteroid Receptors 33 2. Stevens B, Alvarez CM, Bohman R, O’Connor JD: An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells. Cell 22, 675 (1980). 3. Cherbas L, Yonge CD, Cherbas P, Williams CM: The morphological response of &-H cells to ecdysteroids: Hormonal specificity. Wilhelm Roux’s Arch Dev Biol 189, 1(1980). 4. Chang ES, Yudin AI, Clark WN: Hormone action on a Drosophila cell line. In Vitro 28, 297 (1982). 5. Swiderski R: The Induction of Dopa Decarboxylase Activity by 20-OH-Ecdysone in a Drosophila Cell Line. Ph.D. Thesis, University of California, Los Angeles (1985). 6. Cherbas P, Cherbas L, Williams CM: Induction of acetylcholinesterase activity by 0ecdysone in a Drosophila cell line. Science 197, 275 (1977). 7. Savakis C, Demetri G, Cherbas P: Ecdysteroid-inducible polypeptides in a Drosophila cell line. Cell 22, 665 (1980). 8. Sage BA, Tanis MA, O’Connor JD: Characterization of ecdysteroid receptors in cytosol and naive nuclear preparations of Drosophila K, cells. J Biol Chem 257, 6373 (1982). 9. Fristrom JW, Yund MA: Characteristics of the action of ecdysones on Drosophila imaginal discs cultured in vitro. In: Invertebrate Tissue Culture. Maramorosch K, ed. Academic Press, New York, pp 161-178 (1976). 10. Yund MA, King DS, Fristrom JW: Ecdysteroid receptors in imaginal discs of Drosophila rnelanogasfer. Proc Natl Acad Sci USA 75, 6039 (1978). 11. Payvar F, Wrange 0, Carlstedt-Duke J, Okret S, Gastafsson JA, Yamamoto KR: Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment whose transcription is regulated by glucocorticoids in vivo. Proc Natl Acad Sci USA 78, 6628 (1981). 12. Clark JH, Upchurch S, Markaverich B, Eriksson H, Hardin JW: Estrogen receptor heterogeneity and uterotropic response. In: Gene Regulation by Steroid Hormones. Roy AK, Clark JH, eds. Springer-Verlag, New York, pp 89-105 (1980). 13. Taylor CA, Smith HE, Danzo BJ: Photoaffinity labeling of rat androgen binding protein. Proc Natl Acad Sci USA 77, 234 (1980). 14. Dure LS, Schrader WT, O’Malley BW: Covalent attachment of a progestational steroid to chick oviduct progesterone receptor by photoaffinity labelling. Nature 283, 784 (1980). 15. Westphal HM, Fleischmann G, Beato M: Photoaffinity labeling of steroid binding proteins with unmodified ligands. Eur J Biochem 129, 101 (1981). 16. Canonica L, Danieli B, Ferrari G, Haimova MA, Krepinsky J: The structure of a new phytoecdysone kaladasterone: An application of I3C magnetic resonance spectroscopy to structural problems. Experientia 29, 1062 (1973). 17. Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51, 660 (1949).